RESUMO
Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.
Assuntos
Hemicentrotus/embriologia , Hemicentrotus/enzimologia , Esqueleto , Sulfatases/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Clonagem Molecular , Embrião não Mamífero , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Hemicentrotus/genética , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , RNA Mensageiro , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sulfatases/química , Sulfatases/metabolismoRESUMO
MicroRNAs are single-stranded RNA molecules with a length of 19-25 nucleotides, which play roles in various biological phenomena, including development, differentiation, apoptosis, by regulating target gene expression. Although the presence of microRNA molecules in sea urchin and the expression of genes involved in microRNA biogenesis during sea urchin development have been reported recently, the function of microRNA in sea urchin development remains to be elucidated. In this study, to understand the function of microRNA in the early development of sea urchin, we focused on Dicer, an essential enzyme for biosynthesis of mature microRNA. We determined the nucleotide sequence of cDNA for a Dicer homolog in the sea urchin, Hemicentrotus pulcherrimus, HpDcr, and found that functional domains of Dicer proteins are conserved in HpDcr. Analyses of its pattern of expression showed that HpDcr mRNA is expressed in embryos at all developmental stages analyzed, and seems to distribute asymmetrically at the morula and later stages. Knockdown of HpDcr resulted in anomalous morphogenesis, such as impairment of gastrulation and skeletogenesis at the mesenchyme blastula stage and later stages, and alteration of mRNA levels of cell type-specific genes. Thus, HpDcr plays important roles in morphogenesis in sea urchin embryos, suggesting that miRNA could be involved in the early development of sea urchin by regulating target gene expression.
